Cell adhesion (i.e., a process by which cells associate with each other, migrate towards a specific target, or localize within the extracellular matrix) underlies many biological phenomena. Cell adhesion causes adhesion of hemoatopoietic to endothelial cells and the subsequent migration of those hemopoietic cells out of blood vessels and to the site of injury, thus playing a role in mammalian pathologies such as inflammation and immune reactions.
Various cell-surface macromolecules (known as cell adhesion receptors) mediate cell-cell and cell-matrix interactions. For example, the integrins are the key mediators in adhesive interactions between hematopoietic and other cells. Integrins are non-covalent heterodimeric complexes consisting of two subunits, xcex1 and xcex2. Depending on the type of its xcex1 and xcex2 subunit components, each integrin molecule is categorized into its own subfamily. There are at least 12 different xcex1 subunits (xcex11-xcex16, xcex1-L, xcex1-M, xcex1-X, xcex1-IIB, xcex1-V, and xcex1-E) and at least 9 different xcex2 subunits (xcex21-xcex29).
The very late antigen-4 (VLA-4), also known as xcex14xcex21 integrin or CD49d/CD29, is a leukocyte cell surface receptor that participates in a variety of cell-cell and cell-matrix adhesions. It is a receptor for both the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-1 (VCAM-1), and the extracellular matrix protein fibronectin (FN). Anti-VLA-4 monoclonal antibodies (mAb""s) inhibit VLA-4-dependent adhesive interactions both in vitro and in vivo. This inhibition of VLA-4-dependent cell adhesion may prevent or inhibit several inflammatory and autoimmune pathologies.
WO 96/22966 describes compounds of the formula 
as useful for inhibition, prevention, and suppression of VLA-4-mediated cell adhesion.
This invention relates to organic compounds which are VLA-4 antagonists, the preparation of such compounds and their use as pharmaceuticals.
It has now been found that certain novel compounds have very good VLA-4 antagonistic activity and useful pharmacological properties.
Accordingly, the present invention provides in one aspect compounds of formula I 
wherein
R1 is alkyl, alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl, cycloalkenyl, aryl, aryl-substituted alkyl (aralkyl), aryl-substituted alkenyl or alkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl, biaryl, alkoxy, alkenoxy, alkynoxy, aryl-substituted alkoxy (aralkoxy), aryl-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino or alkynylamino, aryl-substituted alkylamino, aryl-substituted alkenylamino or alkynylamino, aryloxy, arylamino, N-alkylureido-substituted alkyl, N-arylureido-substituted alkyl, alkylcarbonylamino-substituted alkyl, aminocarbonyl-substituted alkyl, heterocyclyl, heterocyclyl-substituted alkyl, heterocyclyl-substituted amino, carboxyalkyl substituted aralkyl, oxocarbocyclyl-fused aryl, or heterocyclylalkyl;
R2 is (CH2)qxe2x80x94Vxe2x80x94(CH2)qxe2x80x2xe2x80x94Vrxe2x80x94Rs;
R3 is H, alkyl, alkenyl, aryl, or heteroaryl;
R4 is H, aryl, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl and aryl-substituted alkyl, heterocyclyl, heterocyclylcarbonyl, aminocarbonyl, amido, mono- or dialkylaminocarbonyl, mono- or diarylaminocarbonyl, alkylarylaminocarbonyl, diarylaminocarbonyl, mono- or diacylaminocarbonyl, aromatic or aliphatic acyl, or alkyl optionally substituted by substituents selected from the group consisting of amino, halo, hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy, and heterocyclyl;
R5 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aryl-substituted alkyl, aryl-substituted alkenyl, or alkynyl; alkyl optionally substituted by substituents selected from the group consisting of amino, halo, hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy, and heterocyclyl;
R6 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl-substituted alkenyl or alkynyl, hydroxy-substituted alkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substituted alkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl, alkylsulfonyl-substituted alkyl, (hydroxy-substituted alkylthio)-substituted alkyl, thioalkoxy-substituted alkyl, acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substituted alkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl, thiomorpholinocarbonyl-substituted alkyl, [N-(alkyl, alkenyl or alkynyl)- or (N,N-dialkyl, dialkenyl or dialkynyl)-amino] carbonyl-substituted alkyl, carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl; or amino acid side chains selected from arginine, asparagine, glutamine, S-methyl cysteine, methionine and corresponding sulfoxide and sulfone derivatives thereof, glycine, leucine, isoleucine, allo-isoleucine, tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline, alanine, ornithine, histidine, glutamine, valine, threonine, serine, aspartic acid, beta-cyanoalanine, and allothreonine;
R7 and R8 are independently H, alkyl, alkenyl, carbocyclic aryl, heteroaryl, or alkyl, alkenyl, carbocyclic aryl or heteroaryl substituted by 1-3 substituents selected from the group consisting of amino, hydroxy, mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino, diarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy, thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy, and heterocyclyl;
or R2 and R6 taken together with the atoms to which they are attached may form a heterocycle;
V is O, NH, S, SO, or SO2;
X is CO2R5, PO3H, SO2R5, SO3H, OPO3H, CO2H, or CON(R4)2;
W is CH or N;
Y is CO, SO2, or PO2;
Z is (CH2)nxe2x80x2, CHR6, or NR7;
n and nxe2x80x2 are independently 0-4;
m is 1-4;
p is 1-4;
q and qxe2x80x2 are independently 1-5; and
r is 0 or 1;
or pharmaceutically acceptable salts thereof.
Compounds of the invention, i.e. compounds of formula I and their pharmaceutically acceptable salts, are VLA-4 antagonists and useful to prevent, suppress, or inhibit cell adhesions. Thus, they are useful in VLA-4-mediated cell adhesion disease states, particularly inflammation and autoimmune diseases. They are particularly useful in surgery-induced inflammation, especially transplant surgery. The compounds of the invention may be used alone or in combination with other agents active in the prevention, suppression, or inhibition of cell adhesion.
Another embodiment of the invention is a pharmaceutical composition, particularly a composition for VLA-4 antagonism, comprising an effective amount of a compound of the invention, optionally together with a pharmaceutically acceptable carrier.
In another aspect, the present invention also provides compounds of the invention, i.e. compounds of formula I or pharmaceutically acceptable salts thereof, for use as pharmaceuticals, particularly in VLA-4 antagonism.
In a further aspect the invention provides a method of antagonizing VLA-4 in a mammal which comprises administering to a mammal, preferably man, in need of such treatment an effective amount of a compound of the invention.
In a yet further aspect, the invention provides the use of a compound of the invention for the preparation of a medicament for the treatment of a disease mediated by VLA-4.
Particular embodiments of the invention relate to compounds of formula I or pharmaceutically acceptable salts thereof wherein
(a) R1 is aryl, particularly N-arylureido-substituted phenyl;
(b) R4 is H, alkyl, alkenyl or aryl;
(c) W is CH;
(d) Y is CO;
(e) X is CO2H or CO2alkyl;
(f) Z is (CH2)nxe2x80x2 or CHR6.
Preferred compounds of the invention are those of formula Ia 
wherein
R2 is C1-4alkyl-oxy-C1-8alkyl;
R4 is H, alkyl, alkenyl, carbocyclic aryl or heteroaryl;
X is CO2H or CO2alkyl;
and the other symbols are as defined for formula I; or pharmaceutically acceptable salts thereof.
More-preferred compounds of the invention are those of formula Ia wherein R1 is aryl; R2 is methoxy-n-propyl; R3 is H; R4 is alkenyl or aryl; X is CO2H; n is 0; and W is CH; or pharmaceutically acceptable salts thereof.
A particular embodiment of the invention is directed to compounds of formula Ib 
wherein
R1 is N-arylureidophenyl;
R2 is C1-C4-alkyl-oxy-C2-C4-alkyl;
R3 is H;
R4 is H, C1-C4-alkyl, C2-C4-alkenyl or carbocyclic aryl;
n is 1 or 2;
m is 1, 2 or 3;
X is COOH or CO2R5; and
R5 is optionally substituted lower alkyl;
or pharmaceutically acceptable salts thereof.
Preferred are the compounds of formula Ib wherein
R1 is N-(optionally substituted phenyl)-ureidophenyl;
R2 is methoxypropyl;
R3 is H;
R4 is C2-C4-alkenyl or optionally substituted phenyl;
n is 1;
m is 1; and
X is COOH;
or pharmaceutically acceptable salts thereof.
Another particular embodiment of the invention is directed to compounds of formula Ic 
wherein
Ra is H, CH3, Cl or NH2;
R2 is (CH2)3OCH3 or (CH2)4OCH3;
R4 is xe2x80x94(CH)xe2x95x90(CH)xe2x80x94CH3, phenyl, 4-methoxyphenyl, or 3,4-dimethoxyphenyl; and T is NH or CH2;
or pharmaceutically acceptable salts thereof.
Most-preferred compounds of the invention are those of formula Id 
wherein
R4 is xe2x80x94(CH)xe2x95x90(CH)xe2x80x94CH3, phenyl, 4-methoxyphenyl, or 3,4-dimethoxyphenyl;
or the pharmaceutically acceptable salts thereof.
xe2x80x9cAlkylxe2x80x9d means a straight-chain or branched-chain alkyl radical containing from 1 to 10, preferably from 1 to 6, and more preferably from 1 to 4, carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, and decyl.
xe2x80x9cAlkenylxe2x80x9d means a straight-chain or branched-chain alkenyl radical containing from 2 to 10, preferably from 2 to 6, and more preferably from 2 to 4, carbon atoms. Examples of such radicals include etheryl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, and decenyl.
xe2x80x9cLowerxe2x80x9d in conjunction with the above terms means a said radical containing up to 6 carbon atoms. xe2x80x9cSubstitutedxe2x80x9d in conjunction with the above terms means a said radical substituted by e.g. amino, halo, hydroxy, mercapto, mono- or dialkylamino, mono- or di-arylalkylamino, mono- or diarylamino, alkoxy, aryloxy, aryl, thioaryloxy, thioalkoxy or heterocyclyl.
xe2x80x9cAlkynylxe2x80x9d means a straight-chain or branched-chain alkynyl radical containing from 2 to 10, preferably from 2 to 6, and more preferably from 2 to 4, carbon atoms. Examples of such radicals include ethynyl (acetylenyl), propynyl, propargyl, butynyl, hexynyl, and decynyl.
xe2x80x9cCycloalkylxe2x80x9d means a cyclic alkyl radical containing from 3 to 8, preferably from 3 to 6, carbon atoms. Examples of such cycloalkyl radicals include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclopropyl methyl.
xe2x80x9cCycloalkenylxe2x80x9d means a cyclic carbocycle containing from 4 to 8, preferably 5 to 6, carbon atoms and one or more double bonds. Examples of such cycloalkenyl radicals include cyclopentenyl, cyclohexenyl, cyclopentadienyl, and 2-methyl-2-butenyl.
Aryl means carbocyclic or heterocyclic aryl (heteroaryl).
xe2x80x9cArylxe2x80x9d (carbocyclic aryl and heteroaryl) means a 5- or 6-membered carbocyclic aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N, and S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, and S; or a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, and S; each of which rings is optionally substituted with 1-3 substituents selected from e.g. lower alkyl, alkenyl, alkynyl, substituted lower alkyl, substituted alkenyl, substituted alkynyl, xe2x95x90O, NO2, halogen, hydroxy, alkoxy, cyano, xe2x80x94NRxe2x80x2Rxe2x80x2, acylamino, phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy, wherein each of said phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy is optionally substituted with 1-3 substituents selected from e.g. lower alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, cyano, phenyl, phenoxy, benzyl, benzyloxy, carboxy, carboalkoxy, carboxamido, heteroaryl, heteroaryloxy, NO2, and xe2x80x94NRxe2x80x2Rxe2x80x2, wherein Rxe2x80x2 is H or lower alkyl. The carbocyclic aromatic ring systems comprise phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl. The heterocyclic aromatic ring systems comprise furyl, thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzo[b]thiophenyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.
xe2x80x9cArylxe2x80x9d, as it relates in particular to the grouping R1 in the above formulae, means carbocyclic or heterocyclic aryl, particularly phenyl optionally substituted by one to three substituents which are independently selected from e.g. halo, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano, carboxy, carboalkoxy, Arxe2x80x2-substituted alkyl, Arxe2x80x2-substituted alkenyl or alkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy or alkynoxy, Arxe2x80x2-substituted alkoxy, Arxe2x80x2-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino or alkynylamino, Arxe2x80x2-substituted alkylamino, Arxe2x80x2-substituted alkenylamino or alkynylamino, Arxe2x80x2-substituted carbonyloxy, alkylcarbonyloxy, aliphatic or aromatic acyl such as alkanoyl, Arxe2x80x2-substituted alkanoyl or Arxe2x80x2-substituted carbonyl, Arxe2x80x2-substituted alkylcarbonyloxy, Arxe2x80x2-substituted carbonylamino, Arxe2x80x2-substituted amino, Arxe2x80x2-substituted oxy, alkylcarbonylamino, Arxe2x80x2-substituted alkylcarbonylamino, Arxe2x80x2-substituted aminocarbonylalkyl, alkoxy-carbonylamino, Arxe2x80x2-substituted alkoxycarbonyl-amino, Arxe2x80x2-oxycarbonylamino, alkylsulfonylamino, mono- or bis-(Arxe2x80x2-sulfonyl) amino, Arxe2x80x2-substituted alkyl-sulfonylamino, morpholinocarbonylamino, thiomorpholinocarbonylamino, N-alkyl guanidino, N-Arxe2x80x2 guanidino, N-Arxe2x80x2 cyano-guanidino, N-N-(Arxe2x80x2-, alkyl) guanidino, N,N-(Arxe2x80x2, Arxe2x80x2) guanidino, N,N-dialkyl guanidino, N,N,N-trialkyl guanidino, N-alkyl-ureido, N,N-dialkyl-ureido, N-Arxe2x80x2-ureido, N,N-(Arxe2x80x2, alkyl) ureido and N,N-(Arxe2x80x2)2 ureido; acylcarbonylamino; Arxe2x80x2-substituted aryl; aromatic acyl-substituted aromatic or aliphatic acyl; Arxe2x80x2-substituted heterocyclyl;xe2x80x2 Arxe2x80x2-substituted cycloalkyl or cycloalkenyl; heterocyclylalkoxy; N,N-(Arxe2x80x2, hydroxyl) ureido; Arxe2x80x2-substituted cycloalkyl and cycloalkenyl; Arxe2x80x2-substituted biaryl; Arxe2x80x2-substituted aminocarbonylamino; Arxe2x80x2-mercapto-substituted alkyl; Arxe2x80x2-amino-substituted aryl; Arxe2x80x2-oxy-substituted alkyl; Arxe2x80x2-substituted aminocycloalkyl and cycloalkenyl; aralkylaminosulfonyl; aralkoxyalkyl; N-Arxe2x80x2-substituted thioureido; N-aralkoxyureido; N-hydroxylureido; N-alkenylureido; N,N-(alkyl, hydroxyl)ureido; heterocyclyl; thioaryloxy-substituted aryl; N,N-(aryl,alkyl)hydrazino; Arxe2x80x2-substituted sulfonylheterocyclyl; aralkyl-substituted heterocyclyl; cycloalkyl and cycloalkenyl-substituted heterocyclyl; cycloalkyl-fused aryl; aryloxy-substituted alkyl; heterocyclylamino; Arxe2x80x2-substituted arylaminosulfonyl; Arxe2x80x2-substituted alkenoyl; aliphatic or aromatic acylaminocarbonyl; aliphatic or aromatic acyl-substituted alkenyl; Arxe2x80x2-substituted aminocarbonyloxy; Arxe2x80x2, Arxe2x80x2-disubstituted aryl; aliphatic or aromatic acyl-substituted acyl; benzofused-heterocyclylcarbonylamino; Arxe2x80x2-substituted hydrazino; Arxe2x80x2-substituted aminosulfonyl; Arxe2x80x2-substituted alkylamino; Arxe2x80x2-subtituted heterocyclyl; Arxe2x80x2,Arxe2x80x2disubstituted alkanoylamino; Arxe2x80x2-substituted cycloalkanoylamino; heterocyclylalkoxy; N,N-Arxe2x80x2,hydroxylureido; N,Nxe2x80x2-Arxe2x80x2,hydroxylureido; heterocyclylcarbonylamino; Arxe2x80x2-subtituted aminocarbonylheterocyclyl; Arxe2x80x2-subtituted aminocarbonyl; Arxe2x80x2-subtituted carbonylamino; Arxe2x80x2-subtituted aminosulfonylamino; Arxe2x80x2-subtituted mercaptoalkyl; Arxe2x80x2-amino substituted biaryl; aralkylaminoalkoxy; alkyl- and aryloxy-substituted alkoxy; heterocyclylcarbonyl; Arxe2x80x2-substituted sulfonylalkyl; Arxe2x80x2-amino carbocyclyl; aralkylsulfonyl; aryl-substituted alkenyl; heterocyclylalkylamino; heterocyclylalkylaminocarbonyl; Arxe2x80x2-substituted sulfonylaminoalkyl; Arxe2x80x2-substituted cycloalkyl; thioaryloxyalkyl; thioaryloxymercapto; cycloalkylcarbonylalkyl; cycloalkyl-substituted amino; Arxe2x80x2-substituted arylamino; aryloxycarbonylalkyl; phosphorodiamidyl acid or ester; aryloxydimethylsiloxy; 1,3-indandionylcarbonylalkyl; 1,3-indandionylcarbonyl; oxamidyl; heterocyclylalkylidenyl; formamidinyl; benzalizinyl; benzalhydrazino; arylsulfonylureido; benzilylamino; 4-(N-2-carboxyalkyl-1-(1,3-benzodioxol-5-yl)-amino-N-leucinylalkylamidylarylurea); Arxe2x80x2-carbamoyloxy and alkyl- and aryloxy-substituted ureido; wherein xe2x80x9cArxe2x80x2xe2x80x9d is a carbocyclic or heterocyclic aryl group as defined above having one to three substituents selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylamino or alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl, alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl or N,N-dialkylureido.
xe2x80x9cAlkoxyxe2x80x9d means an alkyl ether radical. Examples of alkyl ether radicals include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy. xe2x80x9cAlkenoxyxe2x80x9d means a radical of formula alkenyl-Oxe2x80x94, provided that the radical is not an enol ether. Examples of alkenoxy radicals include allyloxy and E- and Z-3-methyl-2-propenoxy. xe2x80x9cAlkynyloxyxe2x80x9d means a radical of formula alkynyl-Oxe2x80x94, provided that the radical is not an ynol ether. Examples of alkynoxy radicals include propargyloxy and 2-butynyloxy. xe2x80x9cThioalkoxyxe2x80x9d means a thioether radical of formula alkyl-Sxe2x80x94. xe2x80x9cAlkylaminoxe2x80x9d means a mono- or di-alkyl-substituted amino radical (i.e., a radical of formula alkyl-NHxe2x80x94 or (alkyl)2xe2x80x94Nxe2x80x94). Examples of alkylamino radicals include methylamino, ethylamino, propylamino, isopropylamino, t-butylamino, and N,N-diethylamino. xe2x80x9cAlkenylaminoxe2x80x9d means a radical of formula alkenyl-NHxe2x80x94 or (alkenyl)2Nxe2x80x94, provided that the radical is not an enamine. An example of an alkenylamino radical is the allylamino radical. xe2x80x9cAlkynylaminoxe2x80x9d means a radical of formula alkynyl-NHxe2x80x94or (alkynyl)2Nxe2x80x94, provided that the radical is not an ynamine. An example of an alkynylamino radical is the propargyl amino radical. xe2x80x9cAryloxyxe2x80x9d means a radical of formula aryl-Oxe2x80x94. Examples of aryloxy radicals include phenoxy, naphthoxy, and pyridyloxy. xe2x80x9cArylaminoxe2x80x9d means a radical of formula aryl-NHxe2x80x94. Examples of arylamino radicals include phenylamino (anilido), naphthylamino, 2-, 3- or 4-pyridylamino. xe2x80x9cBiarylxe2x80x9d means a radical of formula aryl-aryl-. xe2x80x9cThioarylxe2x80x9d means a radical of formula aryl-Sxe2x80x94. An example of a thioaryl radical is the thiophenyl radical. xe2x80x9cAryl-fused cycloalkylxe2x80x9d means a cycloalkyl radical which shares two adjacent atoms with an aryl radical. An example of an aryl-fused cycloalkyl radical is the benzofused cyclobutyl radical. xe2x80x9cAliphatic acylxe2x80x9d means a radical of the formula alkyl-COxe2x80x94, alkenyl-COxe2x80x94, or alkynyl-COxe2x80x94derived from a carboxylic acid. Examples of aliphatic acyl radicals include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, acryloyl, crotyl, propiolyl, and methylpropiolyl. xe2x80x9cAromatic acylxe2x80x9d means a radical of the formula aryl-COxe2x80x94. Examples of aromatic acyl radicals include benzoyl, 4-halobenzoyl, 4-carboxybenzoyl, naphthoyl, and pyridylcarbonyl. xe2x80x9cMorpholinocarbonylxe2x80x9d and xe2x80x9cthiomorpholinocarbonylxe2x80x9d mean an N-carbonylated morpholino and an N-carbonylated thiomorpholino radical, respectively. xe2x80x9cAlkylcarbonylaminoxe2x80x9d means a radical of formula alkyl-CONHxe2x80x94. xe2x80x9cAlkylsulfonylaminoxe2x80x9d means a radical of formula alkyl-SO2NHxe2x80x94. xe2x80x9cArylsulfonylaminoxe2x80x9d means a radical of formula aryl-SO2NHxe2x80x94. xe2x80x9cN-alkylureaxe2x80x9d or xe2x80x9cN-alkylureidoxe2x80x9d means a radical of formula alkyl-NHxe2x80x94COxe2x80x94NHxe2x80x94. xe2x80x9cN-arylureaxe2x80x9d or xe2x80x9cN-arylureidoxe2x80x9d means a radical of formula aryl-NHxe2x80x94COxe2x80x94NHxe2x80x94. xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d means fluoro, chloro, bromo, and iodo. xe2x80x9cHeterocyclexe2x80x9d, unless otherwise defined herein, means a stable 3-7 membered monocyclic heterocyclic ring or an 8-11 membered bicyclic heterocyclic ring which is saturated or unsaturated, and which may be optionally benzofused. Each heterocycle consists of one or more carbon atoms and from one to four heteroatoms selected from nitrogen, oxygen, and sulfur, any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen. Any ring nitrogen may be optionally substituted with a substituent R4, as defined herein for compounds of formula I. A heterocycle may be attached at any endocyclic carbon or heteroatom which results in the creation of a stable structure. Preferred heterocycles include 5-7 membered monocyclic heterocycles and 8-10 membered bicyclic heterocycles. Heterocycles may be optionally oxo-substituted at 1-3 ring positions and may optionally be independently substituted with 1-4 aryl substituents. Included are heteroaryl groups as defined herein and saturated heterocycles such as piperidine, morpholine, pyrrolidine, thiazolidine, piperazine and the like.
It is intended that the definitions of any substituent or symbol in a particular molecule be independent of its definitions elsewhere in the molecule. Thus, for example, xe2x80x94N(R4)2 represents xe2x80x94NH2, xe2x80x94NHCH3, xe2x80x94N(CH3)2 etc.
Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R) or (S), or as (D) or (L) for amino acids. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active (R) and (S), or (D) and (L), isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.
In a preferred group of compounds of the invention, where W in formula I is CH, the stereochemistry at this carbon atom is (S), i.e. the compounds are of formula 
where R1, R2, R3, R4, X, Y, Z, m, n and p are as defined for formula I, and their pharmaceutically acceptable salts.
The pharmaceutical compositions of the present invention comprise a compound of Formula I or a pharmaceutically acceptable salt thereof as an active ingredient, and may also contain a pharmaceutically acceptable carrier and, optionally, other therapeutic ingredients. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including organic and inorganic acids or bases.
When a compound of the present invention is acidic, salts may be prepared from pharmaceutically acceptable non-toxic bases. Salts derived from all stable forms of inorganic bases include aluminum, ammonium, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium, zinc, etc. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins such as arginine, betaine, caffeine, choline, N,Nxe2x80x2-dibenzylethylenediamine, diethylamine, 2-diethyl-aminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc.
When a compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, etc. Particularly preferred are citric, hydrobromic, maleic, phosphoric, sulfuric, and tartaric acids. Base salts also include ammonium, alkali metal, and alkaline earth metal salts, salts with organic bases, such as dicyclohexylamine salts, and salts with amino acids such as arginine and lysine. Also, basic nitrigen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl chloride, dialkyl sulfates, such as dimethyl, sulfates, long chain halides such as stearyl chlorides, and aralkyl halides, such as benzyl chlorides.
The compounds of the invention are particularly useful in mammals as VLA-4 antagonists and as inhibitors of VLA-4 associated cell adhesion.
The ability of the compounds of formula I to inhibit VLA-4-associated cell adhesions makes them useful for treating, ameliorating, or preventing a variety of inflammatory, immune and autoimmune diseases. Preferably the diseases to be treated with the methods of this invention are selected from respiratory disorders (such as asthma), arthritis, psoriasis, transplantation rejection, multiple sclerosis, type I diabetes, and inflammatory bowel disease, stem cell mobilization and engraphment, and sickle cell anemia. The compounds of formula I are also useful in transplantation surgery; specifically, for the treatment of xenograft and allograft rejection, both chronic and acute.
As to the respiratory diseases, the compounds of the invention are useful as agents for the symptomatic or prophylactic treatment of inflammatory airways diseases. Such diseases include asthma of whatever type or genesis including both intrinsic (non-allergic) asthma and, especially, extrinsic (allergic) asthma. They are useful for the treatment of bronchitic asthma, exercise-induced asthma, occupational asthma, asthma induced following bacterial infection and other non-allergic asthmas. Treatment of asthma is also to be understood as embracing treatment of patients of less than 4 or 5 years of age exhibiting wheezing symptoms, particularly at night and diagnosed or diagnosable as xe2x80x9cwheezy infantsxe2x80x9d.
Prophylactic efficacy in the treatment of asthma may be manifested by reduced frequency or reduced severity of symptomatic attack, improvement in lung function or improved airways hypereactivity. It may be further evidenced by reduced requirement for symptomatic therapy, i.e. therapy for, or intended to restrict or abort, symptomatic attack when it occurs, for example for anti-inflammatory therapy using a corticosteroid.
Other inflammatory airways diseases which may be treated with compounds of the invention include pneumoconiosis (an inflammatory, commonly occupational, disease of the lungs occasioned by repeated inhalation of dusts) including for example aluminosis, asbestosis, chalicosis, siderosis, silicosis, tabacosis and byssinosis.
Further inflammatory airways diseases which may be treated with compounds of the invention include adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) in the exacerbation phase thereof and exacerbation of airways hyperactivity consequent to other drug therapy, e.g. aspirin or b-agonist bronchodilator therapy.
In view of their anti-inflammatory activity, particularly in relation to inhibition of eosinophil activation, compounds of the invention are also useful for the treatment of related disorders of the airways, e.g. eosinophilia, hypereosinophilia, eosinophilic pneumonia, parasitic infestation (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa, eosinophilic granuloma and eosinophil-related disorders affecting the airways caused by drug- reaction.
Compounds of the invention may also be used in the treatment of allergic inflammatory diseases such as allergic rhinitis.
In accordance with the foregoing, the invention includes:
(A) the use of a compound of the invention, i.e. a compound of formula I or a pharmaceutically acceptable salt thereof, as hereinbefore described, for the preparation of a medicament for the treatment of inflammatory, immune or autoimmune diseases, particularly arthritis, transplant rejection or inflammatory airways diseases, especially asthma; and
(B) a method of treating an inflammatory, immune or autoimmune disease, particularly arthritis, transplant rejection or an inflammatory airways disease, especially asthma, which comprises administering to a mammal, particularly a human, in need of such treatment a compound of the invention as hereinbefore described.
The dosage in vitro may range between about 10xe2x88x926 and 10xe2x88x9210 molar concentrations, preferably between about 10xe2x88x927 and 10xe2x88x929 molar concentrations.
The magnitude of the prophylactic or therapeutic dose of the compounds of the invention will vary with the nature and severity of the condition to be treated with the mammal involved and with the particular compound of the invention and its route of administration. In general, the daily dose range lies in the range of 200 to 0.001 mg/kg body weight of a mammal, preferably 50 to 0.05 mg/kg, and most preferably 1.0 to 0.1 mg/kg, in single or divided doses. In some cases, it may be necessary to use doses outside these ranges. When a composition for intravenous administration is employed, a suitable daily dosage range is from about 50 to 0.0005 mg (preferably 20 to 0.01 mg) compound of the invention per kg body weight. When a composition for oral administration is employed, a suitable daily dosage range is from about 20 to 0.001 mg (preferably 10 to 0.01 mg) compound of the invention per kg body weight. When a composition for ophthalmic administration is employed, a suitable daily dosage range is from about 10-0.01% (preferably 5.0-0.5% compound of the invention, typically prepared as a 2.0-0.1% by weight solution or suspension of the compound in an acceptable ophthalmic formulation.
The compounds of the invention may also be used in combination with other pharmaceutically active ingredients. For example, a typical ocular formulation may comprise the compound alone or in combination with a b-adrenergic blocking agent such as timolol maleate or a parasympathomimetic agent such as pilocarpine. When used in combination, the two active ingredients are present in approximately equal parts.
Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of a compound of the invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc. routes may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The pharmaceutical compositions of the present invention comprise a compound of Formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient, and may also contain a pharmaceutically acceptable carrier and, optionally, other therapeutically active ingredients. The invention includes such compositions for use in the treatment of an inflammatory, immune or autoimmune disease, particularly arthritis, transplant rejection or an inflammatory airways disease, especially asthma.
The compositions include compositions suitable for oral, rectal, topical (including transdermal devices, aerosols, creams, ointments, lotions, and dusting powders), parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration; although the most suitable route in any given case will depend largely on the nature and severity of the condition being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
For example, in the treatment of airways diseases, compounds of the invention may be administered orally, for example in tablet form, or by inhalation, for example in aerosol or other atomisable formulations or in dry powder formulations, using an appropriate inhalation device such as those known in the art. For use in the treatment of allergic rhinitis, the compounds of the invention may also be administered intranasally.
A compound of the invention may be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the nature of the preparation desired for administration, i.e., oral, parenteral, etc. In preparing oral dosage forms, any of the usual pharmaceutical media may be used, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (e.g., suspensions, elixirs, and solutions); or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. in the case of oral solid preparations such as powders, capsules, and tablets. Solid oral preparations are preferred over liquid oral preparations. Because of their ease of administration, tablets and capsules are the preferred oral dosage unit form. If desired, capsules may be coated by standard aqueous or non-aqueous techniques.
In addition to the dosage forms described above, the compounds of the invention may be administered by controlled release means and devices.
Pharmaceutical compositions of the present invention suitable for oral administration may be prepared as discrete units such as capsules, cachets, or tablets each containing a predetermined amount of the active ingredient in powder or granular form or as a solution or suspension in an aqueous or nonaqueous liquid or in an oil-in-water or water-in-oil emulsion. Such compositions may be prepared by any of the methods known in the art of pharmacy. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers, finely divided solid carriers, or both and then, if necessary, shaping the product into the desired form. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granule optionally mixed with a binder, lubricant, inert diluent, or surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Ophthalmic inserts are made from compression molded films which are prepared on a Carver Press by subjecting the powdered mixture of active ingredient and HPC to a compression force of 12,000 lb. (gauge) at 149xc2x0 C. for 1-4 min. The film is cooled under pressure by having cold water circulate in the platen. The inserts are then individually cut from the film with a rod-shaped punch. Each insert is placed in a vial, which is then placed in a humidity cabinet (88% relative humidity at 30xc2x0 C.) for 2-4 days. After removal from the cabinet, the vials are capped and then autoclaved at 121xc2x0 C. for 0.5 hr.
The compositions containing a compound of this invention may also comprise an additional agent selected from the group consisting of cortiocosteroids, bronchodilators, antiasthmatics (mast cell stabilizers), anti-inflammatories, antirheumatics, immunosuppressants, antimetabolites, immunonodulators, antipsoriatics, and antidiabetics. Specific compounds include theophylline, sulfasalazine and aminosalicylates (anti-inflammatories); cyclosporin, FK-506, and rapamycin (immunosuppressants); cyclophosphamide and methotrexate (antimetabolites); and interferons (immunomodulators).
The invention includes a compound of the invention as hereinbefore described in inhalable form and an inhalable medicament comprising such a compound in inhalable form optionally together with a pharmaceutically acceptable carrier in inhalable form.
The inhalable form may be, for example, an atomisable composition such as an aerosol comprising the compound of the invention in solution or dispersion in a propellant or a nebulizable composition comprising a dispersion of the compound of the invention in an aqueous, organic or aqueous/organic medium, or a finely divided particulate form comprising the compound of the invention in finely divided form optionally together with a pharmaceutically acceptable carrier in finely divided form.
An aerosol composition suitable for use as the inhalable form may comprise the compound of the invention in solution or dispersion in a propellant, which may be chosen from any of the propellants known in the art. Suitable such propellants include hydrocarbons such as n-propane, n-butane or isobutane or mixtures of two or more such hydrocarbons, and halogen-substituted hydrocarbons, for example fluorine-substituted methanes, ethanes, propanes, butanes, cyclopropanes or cyclobutanes, particularly 1,1,1,2-tetrafluoroethane (HFA134a) and heptafluoropropane (HFA227), or mixtures of two or more such halogen-substituted hydrocarbons. Where the compound of the invention is present in dispersion in the propellant, i.e. where it is present in particulate form dispersed in the propellant, the aerosol composition may also contain a lubricant and a surfactant, which may be chosen from those lubricants and surfactants known in the art. The aerosol composition may contain up to about 5% by weight, for example 0.002 to 5%, 0.01 to 3%, 0.015 to 2%, 0.1 to 2%, 0.5 to 2% or 0.5 to 1%, by weight of the compound of the invention, based on the weight of the propellant. Where present, the lubricant and surfactant may be in an amount up to 5% and 0.5% respectively by weight of the aerosol composition. The aerosol composition may also contain ethanol as co-solvent in an amount up to 30% by weight of the composition, particularly for administration from a pressurised metered dose inhalation device.
A finely divided particulate form, i.e. a dry powder, suitable for use as the inhalable form may comprise the compound of the invention in finely divided particulate form, optionally together with a finely divided particulate carrier, which may be chosen from materials known as carriers in dry powder inhalation compositions, for example saccharides, including monosaccharides, disaccharides and polysaccharides such as arabinose, glucose, fructose, ribose, mannose, sucrose, lactose, maltose, starches or dextran. As especially preferred carrier is lactose. The dry powder may be in capsules of gelatin or plastic, or in blisters, for use in a dry powder inhalation device, preferably in dosage units of 5 xcexcg to 40 mg of the active ingredient. Alternatively, the dry powder may be contained as a reservoir in a multi-dose dry powder inhalation device.
In the finely divided particulate form, and in the aerosol composition where the compound of the invention is present in particulate form, the compound of the invention may have an average particle diameter of up to about 10 xcexcm, for example 1 to 5 xcexcm. The particle size of the compound of the invention, and that of a solid carrier where present in dry powder compositions, can be reduced to the desired level by conventional methods, for example by grinding in an air-jet mill, ball mill or vibrator mill, microprecipitation, spray-drying, lyophilisation or recrystallisation from supercritical media.
The inhalable medicament may be administered using an inhalation device suitable for the inhalable form, such devices being well known in the art. Accordingly, the invention also provides a pharmaceutical product comprising a compound of the invention in inhalable form as hereinbefore described in association with an inhalation device. In a further aspect, the invention provides an inhalation device containing a compound of the invention in inhalable form as hereinbefore described.
Where the inhalable form is an aerosol composition, the inhalation device may be an aerosol vial provided with a valve adapted to deliver a metered dose, such as 10 to 100 xcexcl, e.g. 25 to 50 xcexcl, of the composition, i.e. a device known as a metered dose inhaler. Suitable such aerosol vials and procedures for containing within them aerosol compositions under pressure are well known to those skilled in the art of inhalation therapy. Where the inhalable form is a nebulizable aqueous, organic or aqueous/organic dispersion, the inhalation device may be a known nebulizer, for example a conventional pneumatic nebulizer such as an airjet nebulizer, or an ultrasonic nebulizer, which may contain, for example, from 1 to 50 ml, commonly 1 to 10 ml, of the dispersion; or a hand-held nebulizer such as an AERx (ex Aradigm, US) or BINEB (Boehringer Ingelheim) nebulizer which allows much smaller nebulized volumes, e.g. 10 to 100 xcexcl, than conventional nebulizers. Where the inhalable form is the finely divided particulate form, the inhalation device may be, for example, a dry powder inhalation device adapted to deliver dry powder from a capsule or blister containing a dosage unit of the dry powder or a multidose dry powder inhalation device adapted to deliver, for example, 25 mg of dry powder per actuation. Suitable such dry powder inhalation devices are well known.
The activities and VLA-4 specificities of the compounds of this invention may be determined using in vitro and in vivo assays.
The cell adhesion inhibitory activity of these compounds may be measured by determining the concentration of inhibitor required to block the binding of VLA-4-expressing cells to fibronectin-, CS1- or VCAM-I-coated plates. In this assay microtiter wells are coated with either fibronectin (containing the CS-1 sequence) or CS-1 or VCAM-I. If CS-1 is used, it must be conjugated to a carrier protein, such as bovine serum albumin, in order to bind to the wells. Once the wells are coated, varying concentrations of the test compound are then added together with appropriately labeled, VLA-4-expressing cells. Alternatively, the test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells. The cells are allowed to incubate in the wells for at least 30 minutes. Following incubation, the wells are emptied and washed. Inhibition of binding is measured by quantitating the fluorescence or radioactivity bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound. VLA-4-expressing cells that may be utilized in this assay include Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral blood lymophocytes (PBLs). The cells used in this assay may be fluorescently or radioactively labeled.
A direct binding assay may also be employed to quantitate the inhibitory activity of the compounds of this invention. In this assay, a VCAM-IgG fusion protein containing the first two immunoglobin domains of VCAM (D1D2) attached above the hinge region of an IgG1 molecule (VCAM 2D-IgG), is conjugated to a marker enzyme, such as alkaline phosphatase (AP). The synthesis of this VCAM-IgG fusion is described in PCT publication WO 90/13300. The conjugation of that fusion to a marker enzyme is achieved by well known crosslinking methods. The VCAM-IgG enzyme conjugate is then placed in the wells of a multi-well filtration plate, such as that contained in the Millipore Multiscreen Assay System (Millipore Corp., Bedford, Mass.). Varying concentrations of the test inhibitory compound are then added to the wells followed by addition of VLA-4-expressing cells. The cells, compound and VCAM-IgG enzyme conjugate are mixed together and allowed to incubate at room temperature. Following incubation, the wells are vacuum drained, leaving behind the cells and any bound VCAM. Quantitation of bound VCAM is determined by adding an appropriate calorimetric substrate for the enzyme conjugated to VCAM-IgG and determining the amount of reaction cell adhesion inhibitory activity.
Compounds of the Examples have measured ICSO values for VLA-4 binding of an order as low as 1 nanomolar.
In order to assess the VLA-4 inhibitory specificity of the compounds of this invention, assays for other major groups of integrins, i.e., xcex22 and xcex23, as well as other xcex21 integrins, such as VLA-5, VLA-6 and xcex14xcex27 are performed. These assays may be similar to the adhesion inhibition and direct binding assays described above, substituting the appropriate integrin-expressing cell and corresponding ligand. For example, polymorphonuclear cells (PMNs) express xcex22 integrins on their surface and bind to ICAM. xcex23 integrins are involved in platelet aggregation and inhibition may be measured in a standard platelet aggregation assay. VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds to laminin. Compounds of the Examples are found to be selective for VLA-4 versus related integrins.
An in vivo assay which tests the inhibition of contact hypersensitivity in an animal is described in P. L. Chisholm et al., Eur. J. Immunol., vol. 23, pp. 682-688 (1993).
An assay which measures the inhibition of Ascaris antigen-induced late phase airway responses and airway hyperresponsiveness in asthmatic sheep is described in W. M. Abraham et al., J. Clin. Invest., vol. 93, pp. 776-87 (1994).
The compounds of the invention may also be tested in the following assay.
Antigen-induced Pulmonary Eosinophilia in the Mouse
Sensitization of mice: Male B6D2F1/J mice are sensitized by i.p. injection of 0.5 mL alum-precipitated antigen containing 8 xcexcg of ovalbumin (OVA) adsorbed to 2 mg of aluminum hydroxide gel in a saline vehicle. Five days later the mice are given a booster injection with OVA/alum. Control animals are sensitized with alum only. Ten mice are used for each group.
Challenge and drug administration: Mice are placed in a 12xc3x9714xc3x9710 inch plexiglass chamber and exposed to aerosolized OVA (0.5% in saline) for 1 hour at the beginning of the experiment (t=0), and five hours later. Low molecular weight antagonists are dissolved in 2% DMSO and 150 mM TRIS, pH 8.8. A solvent control is included for each experiment. Drugs are administered orally 30 min prior to OVA exposure, and 6 hour after the first OVA exposure.
BAL fluid collection and analysis: Animals are sacrificed by CO2 asphyxiation 24 hour after the first antigen challenge. The tracheas are exposed and cannulated. The lungs are lavaged with 0.6 mL buffer (Hanks buffered saline with 10 mM Hepes, 0.5% BSA and 10 U/mL heparin). The number of eosinophils in the lavage is assessed by counting the total number of leukocytes and the percentage of eosinophils for each sample.
The % inhibition is calculated by the formula:   1  -                    (                              #            ⁢                          xe2x80x83                        ⁢            Eos            ⁢                          xe2x80x83                        ⁢            with            ⁢                          xe2x80x83                        ⁢            drug            ⁢                          xe2x80x83                        ⁢            in            ⁢                          xe2x80x83                        ⁢            OA            ⁢                          xe2x80x83                        ⁢            goup                    -                      #            ⁢                          xe2x80x83                        ⁢            Eos            ⁢                          xe2x80x83                        ⁢            in            ⁢                          xe2x80x83                        ⁢            no            ⁢                          xe2x80x83                        ⁢            OA            ⁢                          xe2x80x83                        ⁢            goup                          )                    (                              #            ⁢            Eos            ⁢                          xe2x80x83                        ⁢            in            ⁢                          xe2x80x83                        ⁢            OA            ⁢                          xe2x80x83                        ⁢            group                    -                      #            ⁢                          xe2x80x83                        ⁢            Eos            ⁢                          xe2x80x83                        ⁢            in            ⁢                          xe2x80x83                        ⁢            no            ⁢                          xe2x80x83                        ⁢            OA            ⁢                          xe2x80x83                        ⁢            group                          )              xc3x97    100    ⁢    %  
where
Eos=average number of eosinophils, OA=challenged and no OA=unchallenged mice.
In this assay, compounds of the Examples administered at a dosage of 30 mg/kg give percentage inhibition of eosinophilia values up to 77%.
The compounds of the invention may be synthesized using known techniques. See, e.g., WO 96/22966, incorporated herein by reference, which teaches the synthesis of analogous compounds. The invention is further defined by reference to the following examples, which are intended to be illustrative and not limiting. For example, representative compounds of formula I wherein W is CH are prepared by reacting a compound of formula II
R1xe2x80x94(CH2)pxe2x80x94Yxe2x80x94OHxe2x80x83xe2x80x83(II)
wherein R1, p and Y have meaning as defined hereinabove, or a reactive functional derivative thereof, with a compound of the formula III 
wherein the carboxyl group is in protected form and wherein R2-R4, Z, n and m have meaning as defined hereinabove, and if desired, converting a compound so obtained to another compound of the invention. The condensation is carried out according to methodology well known in the art for amide formation, e.g. in the presence of a condensing agent such as 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride and a base, such as diisopropylethylamine, in an inert solvent (such as methylene chloride), preferably at room temperature.
The starting materials of formula II, such as optionally substituted phenylureidophenylacetic acids, are in turn known in the art or are prepared according to methods known in the art, e.g. by, for example, condensing a p-aminophenylacetic acid ester with the appropriate aryl isocyanate to obtain the corresponding phenylureidophenylacetic acid ester and hydrolyzing the resulting ester.
The starting materials of formula III are in turn prepared by reacting a compound of the formula IV 
wherein the carboxyl group is in protected form (e.g. as an alkyl ester) and R3, R4 and m have meaning as defined hereinabove, with a compound of the formula V
Lxe2x80x94Zxe2x80x94(CH2)nxe2x80x94COOHxe2x80x83xe2x80x83(V)
preferably as a reactive functional derivative thereof, wherein Z is (CH2)nxe2x80x2, or CHR6, and n, nxe2x80x2 and R6 have meaning as defined hereinabove and L is a leaving group, such as halo or (alkyl or aryl)-sulfonyloxy, in the presence of a base, such as triethylamine, to obtain a compound of the formula VI 
wherein the carboxylic acid is in protected form (e.g. as an alkyl ester), and L, R1, R2 and Z have meaning as defined hereinabove, which is in turn reacted with an amine of the formula IX
R2xe2x80x94NH2xe2x80x83xe2x80x83(VII)
wherein R2 has meaning as defined hereinabove under conditions well-known in the art, to obtain a starting material of formula III in protected form (e.g. as an alkyl ester). Hydrolysis, e.g. with base, such as aqueous lithium hydroxide, gives a starting material of formula III.
As noted above in the cited processes, such may be carried out while, if necessary, temporarily protecting any interfering reactive group(s), and then liberating the resulting compound of the invention. In starting compounds and intermediates which are converted to the compounds of the invention in a manner described herein, functional groups present, such as carboxyl, amino and hydroxy groups, are optionally protected by conventional protecting groups that are a common in preparative organic chemistry. Well-known protecting groups and their introduction are described, for example, in J. F. W. McOmie, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, London, New York, T. W. Greene, xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, Wiley, New York. For example, a hydroxy group is advantageously protected in the form of a benzyl ether which can be cleaved by catalytic hydrogenation to obtain a hydroxy substituted product.
The resulting compounds of formula I wherein X is esterified carboxyl (COOR5) can be converted to the corresponding acids e.g. by hydrolysis according to methods well-known in the art.
The abbreviations used in the following Examples have the indicated meaning: